Oxidative stress is one of the major contributing factors in ethanol (alcohol)-mediated cell and tissue damage. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells/tissues is being produced through direct inhibition of the mitochondrial respiratory chain and induction or activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), NADPH-oxidase, and xanthine oxidase. Despite the well-established roles of ROS/RNS in alcohol-induced cellular dysfunction and injury, the target proteins that are oxidatively-modified by elevated ROS/RNS and their functional alterations are poorly understood. To solve these problems, we recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to positively identify oxidized and/or S-nitrosylated proteins in ethanol-exposed hepatoma cells or animal tissues. [unreadable] [unreadable] During this fiscal year, we collaborated with Dr. Pal Pacher, LPS, in applying our targeted proteomics approach to identify oxidized mitochondrial proteins from mice undergoing ischemia-reperfusion (I/R) injury in the absence and presence of a peroxynitrite scavenger MnTMPyP. Results of liver histology analysis and plasma transaminase activities showed that mouse livers following the I/R procedure were severely damaged in the absence of MnTMPyP. These changes were accompanied with elevated levels of nitrite, 3-nitrotyrosine (3-NT), and iNOS compared to those in sham-operated controls. Pretreatment with MnTMTyP fully restored liver histology with normalized levels of plasma transminases, nitrite, 3-NT, and iNOS. To further identify and study functional alterations, oxidatively-modified mitochondrial proteins from three different groups, were labeled with biotin-NM, purified with streptoavidin-agarose, resolved on 2-D gels, and subjected to protein identification. Mass spectrometric data revealed that many mitochondrial proteins, involved in the respiratory chain, energy supply, cellular defense and intermediary metabolism including fat oxidation, and chaperone activities, were oxidatively-modified in the I/R injured mice without MnTMPyP. For instance, NADH-ubiquinone-oxidoreductase (complex I) and ATP synthase (complex V) were oxidized and their activities inhibited in the absence of MnTMPyP. The level of immunoreactive 3-NT in the immunoprecipiated ATP synthase was only observed in the I/R injured mice without MnTMPyP. The 3-NT band was absent in the sham-operated controls or the I/R injured mice treated with MnTMPyP. These result suggest that nitration of active site Tyr residues of ATP synthase appears responsible for the reversible inactivation of the ATP synthase. Mitochondrial aldehyde dehydrogenase (ALDH2), involved in the metabolism of acetaldehyde and toxic lipid peroxides, was S-nitrosylated, and its activity inhibited. Inactivation of these mitochondrial enzymes could lead to mitochondrial dysfunction and hepatic damage in the I/R injured mice while these changes were prevented in the presence of MnTMPyP. Therefore, the current results not only support earlier reports about the role of ROS/RNS in the I/R injury but also provide the underlying mechanism for those results. We believe that this approach can be used in future studies to identify another beneficial agent against oxidative injury in other organs such as brain, heart, lung and kidney. In collaboration with Dr. Norman Salem Jr, LMBB, we also investigated protective effects of polyunsaturatred fatty acids (PUFA) including docosahexaenoic (DHA:22:6n3) and arachidonic acids (AA:20:4n6) against alcoholic fatty liver and mitochondrial dysfunction in Long Evans rats. Chronic exposure to an alcohol liquid diet containing low but adequate levels of linoleic and linolenic acids without DHA and AA (BASE-EtOH) caused fatty liver determined by histological and biochemical measurements. However, alcoholic fatty liver was prevented in rats fed the DHA-supplemented PUFA diet despite similar levels of ethanol consumed (PUFA-EtOH). The levels of CYP2E1, iNOS, and mitochondrial ROS were elevated in the BASE-EtOH group compared to those in pair-fed rats (BASE-control). These increases were normalized in the rats fed the PUFA-EtOH diet. Targeted proteomics results showed that many mitochondrial proteins were oxidatively-modified in the BASE-EtOH group but normalized in the PUFA-EtOH group. Immunoblot analysis with the antibody against 3-NT showed that Tyr residues of ATP synthase were nitrated in the BASE-EtOH group, resulting in its inhibition and reduced ATP production. The decreased ATP synthase activity was restored and the 3-NT band disappeared in the PUFA-EtOH group. The ALDH2 activity in the BASE-EtOH group was inhibited through S-nitrosylation based on the recovery of its activity after incubation with ascorbate or DTT. However, the suppressed ALDH2 activity was restored in the PUFA-EtOH group. The reversible ALDH2 inactivation positively correlated with the respective level of malondialdehyde in different groups. Furthermore, mitochondrial 3-ketoacylCoA thiolase involved in the beta-oxidation of fatty acids was oxidatively-modified and inactivated in the BASE-EtOH group, compared to that in pair-fed BASE-control rats. The decreased 3-ketoacyl-CoA thiolase activity was normalized in the PUFA-EtOH group. These results suggest that physiologically relevant levels of DHA-containing PUFA are protective against alcohol-mediated mitochondrial dysfunction and fatty liver through decreasing the levels of ROS/RNS. We expect that DHA-supplemented PUFA diet may also provide beneficial effects against non-alcoholic hepatosteatosis caused by obesity, diabetes, and many drugs being used in clinics. Two manuscripts about these results have been prepared and will be submitted for publications soon. [unreadable] [unreadable] Our recent data also showed that cytosolic ALDH1 was oxidatively-modified in the liver from ethanol-exposed rats. Because ALDH1 exhibits a relatively low Km value for acetaldehyde (Km=14-15 M), high levels of acetaldehyde cannot be accumulated in knockout mice deficient of the mitochondrial ALDH2 gene. However, recent data from another laboratory showed that the blood and tissue levels of acetaldehyde were markedly elevated in ethanol-exposed ALDH2 knockout mice, compared with those in wild type mice. Because of the conflicting data, we investigated whether hepatic ALDH1 activity can be inhibited in ethanol-exposed rats. Chronic or binge ethanol exposure significantly decreased the ALDH1 activity, which was restored by addition of DTT. Immunoblot analysis with the anti-S-NO-Cys antibody showed one immunoreactive band in the immunoprecipiated ALDH1 from ethanol-exposed rats, but not from pair-fed controls. Therefore ethanol can inhibit the ALDH1 activity via S-nitrosylation, leading to acetaldehyde accumulation, as observed in ethanol-exposed animals.[unreadable] [unreadable] Finally, we also studied the mechanism of ethanol-mediated apoptosis and the role of stress-activated protein kinases in phosphorylating Bax before its translocation to mitochondria and apoptosis in HT-29 cells. Our unpublished results showed that ethanol activated JNK and p38 kinase, which phosphorylated Bax in HT-29 cells in a time- and dose-dependent manner. Phosphorylated Bax was then translocated to mitochondria and cytochrome c was released from mitochondria. Subsequently, caspase 3 was activated and apoptosis took place in later times. Treatment with specific inhibitors of JNK or p38 kinase inhibited JNK or p38 kinase activity, Bax phosphorylation determined by the pI shift on 2-D gels, and its translocation to mitochondria prior to apoptosis. These results indicated that activation of stress-activated kinases and phosphorylation of Bax play critical roles in ethanol-mediated apoptosis. We are preparing one manuscript about these results.